The live attenuated poliovirus vaccines developed by Sabin in the 1950s have found great use throughout the world. Vaccine strains derived from each of the three poliovirus serotypes, known as Sabin types 1, 2 and 3, were prepared by passage of wild-type viruses in cell cultures and whole animals until attenuated strains were obtained. These attenuated viruses are substantially less able to cause poliomyelitis in humans than the original wild-type strains. They are administered orally and replicate in the gut to induce a protective immune response.
Although the live oral poliovirus vaccines are generally regarded as safe, their use is associated with a small incidence of paralysis in vaccinees. This is most often associated with type 2 and 3 serotypes and rarely, if ever, with type 1. Efforts have, therefore, been made to develop improved type 2 and type 3 vaccines which would be at least comparable in safety to the excellent type 1 strain.
The Sabin vaccine strains were developed by essentially empirical procedures. The genetic basis of their attenuation is not completely understood. Over the past several years, however, scientists have employed a number of molecular biological techniques in an attempt to elucidate the mechanism by which the neurovirulence of these vaccine strains is reduced. Most of the work has concentrated on serotypes 1 and 3. For both of these the complete nucleotide sequences of the vaccine strains have been compared with those of their neurovirulent progenitors.
In the case of poliovirus type 1, the vaccine strain differs from its progenitor at 47 positions in the 7441 base genome (Nomoto et al., Proc. Natl. Acad. Sci. USA 79:5793-5797, 1982). All of these are simple point mutations and 21 of them give rise to amino acid changes in virus-coded proteins. Although several mutations are thought to contribute to the attenuation phenotype of the vaccine strain, direct evidence has been presented that the mutation of A-G at position 480 in the 5′ non-coding region of the genome has a marked attenuating effect on the virus (Nomoto et al., UCLA Symp. Mol. Cell. Biol., New Series, 54 (Eds M. A. Brinton and R. R. Rueckert):437-452, New York: Alan R. Liss Inc., 1987)).
Analogous studies on poliovirus type 3 reveal just 10 nucleotide sequence differences in the 7432 base genome between the vaccine and its progenitor strain (Stanway et al., Proc. Natl. Acad. Sci. USA 81:1539-1543, 1984). Just three of these give rise to amino acid substitutions in virus-encoded proteins. The positions of bases in the 5′ non-coding region of the genome of type 3 poliovirus are numbered herein according to the numbering system of Stanway et al., 1984.
The construction of defined recombinants between the type 3 Sabin vaccine strain and its progenitor strain has allowed the identification of the mutations which contribute to the attenuation phenotype. One of these is at position 2034 and causes a serine to phenylalanine change in virus protein VP3.
The other mutation of interest is C (progenitor) to U (vaccine strain) at position 472 in the 5′ non-coding region of the genome. This 472 U mutation has been observed to revert to the progenitor (wild-type) 472 C rapidly upon replication of the virus in the human gut (Evans et al., Nature 314:548-550, 1985). This reversion is associated with an increase in neurovirulence. C at position 472 has also been shown to be essential for growth of a mouse/human polio recombinant virus in the mouse brain (La Monica et al., J. Virol. 57:515-525, 1986). More recently, it has been observed that A changes to G at position 481 in poliovirus type 2, again upon replication of the virus in the gut of vaccinees (Macadam et al., Virology 181:451-458, 1991).
A model for the secondary structure of the 5′ non-coding region of the genome of poliovirus type 3 Leon strain has previously been proposed (Skinner et al., J. Mol. Biol. 207: 379-392, 1989). As concerns domain V (nucleotides 471-538), bases at positions 471-473 and 477-483 are paired with bases at positions 538-536 and 534-528 respectively as follows:
      471         477       483. . . U C C . . . C C A U G G A . . .. . . A G G . . . G G U G C C U . . .      538         534       528
For convenience, the paired regions are termed stem (a) (471-473/538-536) and stem (b) (477-483/534-528). Previously, we found that a type 3 poliovirus with the base pair 472-537 reversed, i.e. 472 G and 537 C, is attenuated. Further, this attenuated virus had a slightly lower LD50 value than the corresponding poliovirus which only had the mutation C to G at position 472 but which retained the wild-type G at position 537. Attenuated polioviruses in which a base pair of stem (a) or stem (b) of domain V is reversed are disclosed in EP-A-0383433. However, subsequent experiments showed that the type 3 poliovirus in which the 472-537 base pair is reversed is not as attenuated as the type 3 Sabin vaccine strain.
We have also reported previously the production of attenuated polioviruses which have substantially the same attenuation as, or greater attenuation than, the Sabin vaccine strain (so that they are safe to use) but which are much more stable genetically. These attenuated polioviruses do not have a U-G base pair or other base pair mismatch in stem (a) or (b) of domain V of the 5′ non-coding region of the poliovirus genome. (A departure from Watson-Crick base pairing is considered to be a mismatch.) More specifically, we prepared type 3 polioviruses which contained the following U-A base pairs:
(a) S15: U-A at 472-537, U-A at 480-531 and U-A at 481-530; or
(b) S16: U-A at 472-537, U-A at 480-531 and A-U at 482-529.
Under conditions which rapidly selected neurovirulent variants of Sabin 3, the attenuation phenotypes of these poliovirus strains were stable (WO98/41619).
As a result of the success of the global polio eradication programme the proportion of cases attributable to vaccine-derived strains has increased dramatically and will continue to do so until live virus vaccination ceases. Partly in response to this, many developed countries have already switched to inactivated poliovaccines (IPV) which are currently produced from wild strains. When wild-type polio is eradicated wild-type strains will require high levels of biological containment, which may not be easy to reconcile with the production scales required for IPV, making the use of attenuated vaccine strains for IPV manufacture attractive, though it has been argued that both wild and attenuated strains ultimately present the same containment issues.
There remains a need for poliovirus strains that are non-infectious for humans at exposure levels potentially encountered in vaccine production facilities. This would significantly reduce the likelihood of escape into the environment and the consequences of escape would be negligible even after live virus vaccination has ceased. Such strains may be grown under containment levels that are not prohibitive for vaccine manufacturers.